Soybean-nodulating Rhizobium species are classified as R. japonicum. Older literature references to R. japonicum refer to strains characterized as "slow-growing" Rhizobia. More recent studies of biochemical and genetic characteristics have led to reclassification of "slow-growing" Rhizobia in the genus Bradyrhizobium (Jordan, D. C. (1982) Int. J. Syst. Bacteriol. 32:136). Furthermore, certain "fast-growing" strains have been found which are classified as R. japonicum on the basis of their ability to nodulate Glycine Max cv. Peking, an undeveloped Asian cultivar of soybeans. One such "fast-growing" strain, USDA 191, has been found able to form fix.sup.+ (nitrogen fixing) nodules on commercial soybean cultivars, e.g., Williams (Yelton et al. (1983) J. Gen. Microbiol. 129:1537-1547). Since the literature sometimes refers to slow-growing (Bradyrhizobium) strains simply as R. japonicum, confusion may occur. For clarity herein, "slow-growing" commercial soybean nodulating strains, such as USDA 110 or USDA 123, are termed Bradyrhizobium japonicum strains, while USDA 191, a "fast-growing" strain, is termed a Rhizobium japonicum strain. R. japonicum USDA 191 is much more, amenable to genetic manipulation than B. japonicum strains because it grows faster, many of the primary genes for symbiosis are located on a plasmid (Appelbaum et al. (1985) J. Bact. 163:385) and transposon mutagenesis occurs at higher frequencies in R. japonicum than in B. japonicum.
The interaction between a Rhizobium strain and a leguminous plant to form a nitrogen fixing root nodule involves the participation of a number of bacterial genes which in many Rhizobium strains are located on a large plasmid known as the Sym plasmid. (For a recent review see Long, S. R. (1984) in Plant-Microbe Interactions, T. Kosuge and E. Nester, eds., McMillan, New York, pp. 256-306.) Analysis of USDA 191 plasmids by gel electrophoresis has revealed the presence of several plasmids including a 200 MD plasmid which hybridizes to nif and nod probes from Klebsiella pneumoniae and Rhizobium meliloti and has been designated pSym 191 (Appelbaum et al. (1985)). Genetic loci involved in nodulation, designated nod, have been cloned from the Sym plasmid DNA of several Rhizobium species (Long et al. (1982) Nature 298:485) (R. meliloti); Downie, J. A. et al. (1983) EMBO J. 2:947 (R. leguminosarum); Schofield, P. R. et al. (1983) Mol. Gen. Genet. 192:459 (R. trifolii); Kondorosi, E. et al. (1984) Mol. Gen. Genet. 193:445 (R. meliloti). A set of nod genes, designated nodA, nodB, nodC and nodD, have been identified in each case, which appear to be involved in early stages of infection and nodule development. Mutants in these genes fail to develop visible nodules and in some cases fail to display root hair curling which is believed to be an initial reaction in the nodulation process. These genes have been termed "common nod genes" because they appear to be functionally interconvertible between the species R. meliloti, R. trifolii and R. leguminosarum, as shown by interspecific complementation experiments of nod mutants by Sym plasmids or by cloned nod genes. The nucleotide sequence of the nodD gene of R. meliloti has been published by Egelhoff, T. T. et al. (1985) DNA 4:241. The sequence yields a predicted amino acid sequence of 308 amino acids which appears to be separately transcribed from nodA, B, and C in R. meliloti. All four genes were closely linked; however, nodD was separately transcribed in R. meliloti.
The function of the nodD protein has not been precisely defined. In R. meliloti, Tn5 insertions in nodD cause a leaky nod.sup.- phenotype; however, in R. trifolii, a Tn5 insertion displayed an unequivocal nod.sup.- phenotype, Schofield et al. (1983). Egelhoff et al. (1985) have suggested the possibility that nodD may be a regulatory gene.
As with any gene coding for a protein, the functional properties of the gene may vary as a result of differences in nucleotide sequence which affect the amino acid sequence of the protein for which it codes, which in turn may affect the functional properties of the protein. Such changes in gene structure may be manifested by differences in the phenotype associated with variants of the gene, differences in complementation behavior, differences in phenotypic behavior in a new genetic background as for example by transfer to a different species of host cell, or by differences in the effects produced by mutation in a gene when compared with its homolog. Such effects are well known in the art and numerous examples are documented in standard texts of molecular genetics, biochemistry or microbial genetics.
The nodulation process in soybeans differs from that in alfalfa, clover or peas in several significant respects. The soybean nodule is anatomically and metabolically different from nodules in the aforementioned legumes, being determinate as compared to the "indeterminate" nodules of the aforementioned legumes. Such differences reflect a different manner of nodule development (Newcomb, W. (1981) in Intl. Rev. Cytol. Suppl. 13 [K. Giles and A. Atherly, eds.] Academic Press N.Y., pp. 247-298). In addition, until recently only species of Bradyrhizobium were known to possess the ability to nodulate soybeans. Whether these differences in the developmental differentiation leading to complete nodule formation are solely the result of genetic differences in soybean, and whether genes of the bacteria which nodulate soybean are specialized to operate in concert with the soybean system, remain open question.